Structure Analysis of Unsaturated Polymyxin E Components Based on High-Performance Liquid Chromatography - Quadrupole/ Time of Flight Tandem Mass Spectrometry and Photochemical Reaction

Background: Polymyxin E (PME), a complex of cationic cyclic lipodecapeptides, is used to treat multidrug-resistant gram-negative bacterial infections. Besides the main components PME1 and PME2, polymyxin containing unsaturated fatty acyl (FA) group with lower contents can hardly determine the structure without chromatographic preparations and NMR. Introduction: The peptide sequences of PME components have been carried out based on highperformance liquid chromatography-quadrupole / time-of-flight mass spectrometry (HPLCQ/ TOF-MS). However, the components with double bonds on the FA, such as 2’, 3’-dehydro PME1, were difficult to be determined or easily misjudged by MS/MS. The transformation of such unsaturated components to be epoxidized or di-hydroxylated components can promote the acquisition of more fragment ions in the MS/MS to assist in judging the position of double bonds on FA. Methods: In this paper, the PME mixtures were dissolved in an equal proportion of 20% ACN aqueous solution and 2-acetylpyridine. The above PME solution was transferred to a quartz cuvette and irradiated with the ultraviolet lamp at 254 nm for 8h. The dehydro PME components were converted to epoxy PMEs and dihydroxy PMEs. A fragmentation pathway of epoxidized or di-hydroxylated components based on Q/TOF-MS/MS was proposed for the first time. Results: According to the characteristic ions of epoxidized components and di-hydroxylated components, 2’, 3’-epoxy PME1/E2 and 2’, 3’-dihydroxy PME1/E2 were confirmed. It can be inferred that the double bond is located at the 2’, 3’-position of FA. Conclusion: The structure of unsaturated PME components with double bonds on the FA is elucidated by HPLC-Q/TOF-MS combined with photochemical reaction. This strategy applies to other lipopeptides containing unsaturated FA chains.